Substantia gelatinosa (SG) neurons in the spinal cord are the principal site of termination of primary afferents, many of which innervate nociceptors. Endogenous opiates or synthetic compounds are thought to produce their anti-nociceptors. Endogenous opiates or synthetic compounds are thought to produce their anti-nociception by interacting with subtypes of opiate receptors on SG neurons. Two recently isolated tetrapeptides endomorphin (Endo) 1 and 2 are believed to be the endogenous ligand for the mu-subtype of opioid receptors. Preliminary results showed endomorphin-like immunoreactivity (Endo-LI) subtype of opioid receptors. Preliminary results showed that endomorphin-like immunoreactivity (Endo-LI) is localized to dense networks of nerve fibers in the superficial layers of the rat dorsal horn. Thus, the rat dorsal horn offers a unique opportunity to test the hypothesis that Endo-1/Endo-2 is released endogenously and that it may modulate the activity of SG neurons. Two major issues will be addressed. First, release of endogenous of endogenous endomorphins will be evaluated in anesthetized rats in vivo or isolated rat spinal cords in vitro by the radioactive microprobe techniques. Electrical stimulation of afferent fibers of painful stimulus to the hindpaw will be employed to evaluate whether or not endomorphin release is altered under these conditions. Second, whole-cell patch recording techniques will be used to study the cellular action and the signal transduction mechanism underlying the action of Endo on single SG neurons in rat transverse spinal cord slices. Our preliminary results show that Endo inhibits the activity of SG neurons by hyperpolarizing the membrane and/or attenuating synaptic transmission. In this proposal, the pre- and post-synaptic actions of Endo will be evaluated electrophysiologically and pharmacologically. The subtype(s) of K+ channels that may underlie the hyperpolarizing action of Endo will be examined. Similarly, the subtype(s) of Ca2+ and/or K+ channels coupled to the presynaptic opiate receptor that may mediate the synaptic depressant action of Endo will be evaluated. The long term goal of this project is to improve our current understanding of the site and mechanism of action of this new class of opioid peptides on dorsal horn neurons, with the aim toward developing a novel class of opiate compounds with therapeutic potentials.